Muscle translational-control RNA (tcRNA) has been separated into two classes, polysomal and messenger ribonuclear protein (mRNA - protein), which have different sizes as determined by acrylamide gel electrophoresis. While normally translation of mRNA - protein mRNA is inhibited by tcRNA derived from the same mRNA - proteins, this inhibition does not occur if the messenger is previously de-adenylated. This suggests that the poly(A) segment of mRNA is required for the tcRNA activity. Utilizing different mRNA - protein fractions from muscle, myosin mRNA - protein and small mRNA - proteins ( less than 30 S), we have been able to demonstrate that a degree of specificity exists in the interaction of tcRNA and mRNA derived from the same mRNA - proteins. This is illustrated by the facts that (a) each tcRNA only inhibits the translation of its respective mRNA and (b) the highest percentage of structural change occurs when each tcRNA is hybridized to its respective mRNA as measured by its resistance to T1 and T2 RNase.

BACKGROUND

The riboprotein telomerase has been linked to cellular immortality and is believed to play a key role in tumorigenesis.

OBJECTIVE

To determine if telomerase is expressed in patients with oral squamous cell carcinoma.

METHODS

Twenty patient samples of oral squamous cell carcinoma and 20 adjacent histologically normal mucosal samples were assayed using the telomeric repeat amplification protocol (TRAP) method for detection of telomerase activity. The leukemic cell line, K562, was used as a positive control and the human fibroblast line, Hs21Fs, as a negative control.

METHODS

Consecutive series of patients with oral squamous cell carcinoma presenting to a tertiary referral center.

METHODS

A sample was classified as telomerase positive when an RNase-sensitive hexameric repeat ladder was observed. Absence of laddering was considered a negative result.

RESULTS

Eighteen (90%) of 20 tumor samples and 7 (35%) of 20 adjacent histologically normal samples were telomerase positive. A statistically significant difference was observed in telomerase activity for T1 and T2 cancers compared with T4 cancers (P<.05 by analysis of variance). No statistically significant difference was observed in activity for T1 and T2 cancers vs T3 cancers.

CONCLUSIONS

The finding of telomerase activity in 90% of tumor samples is consistent with the concept of telomerase playing a key role in tumorigenesis. Further study is needed to determine the usefulness of this enzyme as a molecular marker.

Ribonuclease LE (RNaseLE) from tomato (Lycopersicon esculentum Mill. cv. Lukullus) belongs to the widespread RNase T2 family of ribonucleases. With the exception of S-RNases of the solanaceous self-incompatibility system the functions of other members of the RNase T2 family are only barely understood. Using a 2.6-kbp putative promoter sequence of RNaseLE in front of the uidA reporter gene, expression of beta-glucuronidase in developing phloem tissue and, especially, in the meristematic and elongation zones at root tips was detected. The tissue-specific expression accords with the range of cis-acting elements detected in the RNaseLE promoter. RNaseLE mRNA was localized in developing phloem cells but not in mature phloem tissue, suggesting association of RNaseLE expression with phloem development. Histochemical staining of beta-glucuronidase activity as well as detailed inspection of RNaseLE at mRNA, protein and enzyme activity levels revealed that the wound-induced expression of RNaseLE was also restricted to vascular tissue. RNaseLE transcript accumulation detected by in situ hybridization occurred preferentially in phloem and cambial cells of stem sections upon wounding. The data provide evidence for a role of RNaseLE in a tissue-specific wound response and in wound healing of tomato.

Ribonucleases belonging to the RNase T2 family are enzymes associated with the secretory pathway that are almost absolutely conserved in all eukaryotes. Studies in plants and vertebrates suggest they have an important housekeeping function in rRNA recycling. However, little is known about this family of enzymes in protostomes. We characterized RNase X25, the only RNase T2 enzyme in Drosophila melanogaster. We found that RNase X25 is the major contributor of ribonuclease activity in flies as detected by in gel assays, and has an acidic pH preference. Gene expression analyses showed that the RNase X25 transcript is present in all adult tissues and developmental stages. RNase X25 expression is elevated in response to nutritional stresses; consistent with the hypothesis that this enzyme has a housekeeping role in recycling RNA. A correlation between induction of RNase X25 expression and autophagy was observed. Moreover, induction of gene expression was triggered by oxidative stress suggesting that RNase X25 may have additional roles in stress responses. Phylogenetic analyses of this family in protostomes showed that RNase T2 genes have undergone duplication events followed by divergence in several phyla, including the loss of catalytic residues, and suggest that RNase T2 proteins have acquired novel functions. Among those, it is likely that a role in host immunosuppression evolved independently in several groups, including parasitic Platyhelminthes and parasitoid wasps. The presence of only one RNase T2 gene in the D. melanogaster genome, without any other evident secretory RNase activity detected, makes this organism an ideal system to study the cellular functions of RNase T2 proteins associated with RNA recycling and maintenance of cellular homeostasis. On the other hand, the discovery of gene duplications in several protostome genomes also presents interesting new avenues to study additional biological functions of this ancient family of proteins.

Acinetobacter baumannii is an emerging bacterial pathogen of considerable medical concern. The organism's transmission and ability to cause disease has been associated with its propensity to colonize and form biofilms on abiotic surfaces in health care settings. To better understand the genetic determinants that affect biomaterial attachment, we performed a transposon mutagenesis analysis of abiotic surface-colonization using A. baumannii strain 98-37-09. Disruption of an RNase T2 family gene was found to limit the organism's ability to colonize polystyrene, polypropylene, glass, and stainless steel surfaces. DNA microarray analyses revealed that in comparison to wild type and complemented cells, the RNase T2 family mutant exhibited reduced expression of 29 genes, 15 of which are predicted to be associated with bacterial attachment and surface-associated motility. Motility assays confirmed that RNase T2 mutant displays a severe motility defect. Taken together, our results indicate that the RNase T2 family protein identified in this study is a positive regulator of A. baumannii's ability to colonize inanimate surfaces and motility. Moreover, the enzyme may be an effective target for the intervention of biomaterial colonization, and consequently limit the organism's transmission within the hospital setting.

Ribonucleases (RNases) occur in different gene families, functioning in RNA processing and degradation. In this study, we report on cloning and characterization of RNaseLER, the first class II gene of the RNase T2 family in tomato (Solanum lycopersicum). The family also includes the class I members RNaseLE and RNaseLX, and the class III group of S-RNases acting in self incompatibility. The RNaseLER gene was cloned by polymerase chain reaction (PCR)-assisted methods. Structural analyses of RNaseLER and homologous genes revealed unique key features of class II RNase T2 genes. RNaseLER is a single copy gene in tomato and codes for a primary protein of 260 amino acids. Subcellular localization analyzed with a RNaseLER-eYFP fusion protein and co-localization experiments revealed an intracellular accumulation in the endoplasmic reticulum. Transgenic Nicotiana benthamiana plants carrying the uidA reporter gene under the control of a 900-bp RNaseLER promoter sequence express the reporter gene predominantly in guard cells and trichomes. This previously unknown spatial expression of a RNase T2 gene is consistent with ubiquitous detection of low RNaseLER transcript abundances in almost all parts of tomato plants. As revealed by quantitative real-time RT-PCR analysis treatments with abscisic acid, ethylene or other abiotic and biotic stress factors did not affect RNaseLER expression significantly. Unlike tomato class I genes, RNaseLER represents a constitutively expressed gene with a cell-specific role in stomata and trichomes and no involvement in stress responses.

Schistosome infection typically induces a polarized Th2 type host immune response. As egg antigen molecules play key roles in this immunoregulatory process, clarifying their functions in schistosomiasis would facilitate the development of vaccine and immunotherapeutic methods. Schistosoma japonicum (Sj) CP1412 (GenBank: AY57074.1) has been identified as a new member of the RNase T2 family with immune regulatory functions.

The expression plasmid Sj CP1412-pET2RNase activity of Sj CP1412 was predicted by bioinformatic analysis and confirmed by digesting the yeast tRNA with rSj CP1412.C57BL/6j mice were immunized with rSj CP1412, and its immune regulatory effects in vivo and in vitro were investigated. Meanwhile, the relationship between the RNase activity of Sj CP1412 and its immune regulation was observed.

Sj CP1412 was confirmed as a novel RNase T2 family protein with RNase activity. Immunoblotting and RT-PCR analyses demonstrated Sj CP1412 as a protein exclusively secreted/excreted from eggs, but not cercariae and adult worms. Stimulating RAW264.7 macrophages with rSj CP1412 raised the expression of CD206, Arg-1 and IL-10, which are related to M2 type macrophage differentiation. Stimulating dendritic cells (DCs) with rSjCP1412 failed to induce their maturation, and the recombinant protein also inhibited LPS-stimulated DC maturation. Depletion of Sj CP1412 from soluble egg antigen (SEA) impaired the ability of SEA to induce M2 type polarization of RAW264.7 macrophages. Immunizing mice with rSj CP1412 induced high antibody titers, increased serum IL-4 and TGF-β levels and splenic CD4 + CD25 + Foxp3 + T cells, downregulated serum IFN-γ levels and alleviated the egg granuloma pathology of schistosome infection. In vitro stimulation by rSj CP1412 significantly increased CD4 + CD25 + Foxp3 + T cell numbers in splenocytes of healthy mice. The rSj CP1412 protein with RNase activity inactivated by DEPC failed to induce M2 surface marker CD206 expression in RAW264.7 macrophages.

The Sj CP1412 protein expressed specifically in S. japonicum eggs is a novel member of the RNase T2 family. Similar to Omega-1 of Schistosoma mansoni, the Sj CP1412 protein drives polarization of the host Th2 immune response, which is dependent on its RNase activity. These data provide new evidence towards understanding the immune regulatory role of RNase T2 family proteins during schistosome infection.

We examined the base modification pattern of Xenopus tRNA(Sec) using microinjection into Xenopus oocytes, with particular focus on the wobble base U34 at the first position of the anticodon. We found that U34 becomes modified to mcm5U34 (5-methylcarboxymethyluridine) in the oocyte cytoplasm in a rather complex manner. When the tRNA(Sec) gene is injected into Xenopus oocyte nuclei, psi 55 and m1A58 are readily obtained, but not mcm5U34. This will appear only upon cytoplasmic injection of the gene product arising from the first nuclear injection. In contrast, tRNA(Sec) produced by in vitro transcription with T7 RNA polymerase readily acquires i6A37, psi 55, m1A58, and mcm5U34. The latter is obtained after direct nuclear or cytoplasmic injections. It has been reported by others that mcm5Um, a 2'-O-methylated derivative of mcm5U34, also exists in rat and bovine tRNA(Sec). With both the gene product and the in vitro transcript, and using the sensitive RNase T2 assay, we were unable to detect under our conditions the presence of a dinucleotide carrying mcm5Um and that would be therefore refractory to hydrolysis. We showed that the unusual mcm5U acquisition pathway does not result from impairment of nucleocytoplasmic transport. Rather, these data can be interpreted to mean that the modification is performed by a tRNA(Sec) specific enzyme, limiting in the oocyte cytoplasm.

An rnc70 gene encoding a mutant bacterial ribonuclease III (RNase III) was introduced into wheat (Triticum aestivum cv. Bobwhite) by microprojectile bombardment. T1, T2, and T3 plants regenerated from three transgenic callus lines were challenged with barley stripe mosaic virus. Plants expressing RNase III exhibited a high level of resistance to the virus infection. This resistance was evidenced by the absence of virus symptoms and reduced accumulation of virions in these plants. The result demonstrates that this pathogen-targeted resistance strategy can be effectively employed in conferring resistance to viral diseases of cereal crops.

Two new RNase T2 Ribonucleases, RNase Le37 and Irp3, with a molecular mass of 45 kDa, have been isolated from Basidiomycetes fungi, Lentinus edodes and Irpex lacteus, respectively. The ribonucleases consisted of three domains: an RNase active domain, a Ser/Thr rich domain similar to that of many fungal glycanhydrolases, and a C-terminal 10 kDa domain similar to that of RNase Rny1 in yeast. The locations of hydrophobic amino acids and Pro in the 10 kDa domain of the two basidiomycetous enzymes are very similar to those of RNase Rny1, indicating that these domains may have similar roles.

Analyses were performed on the purified initiator tRNA from rabbit liver to test for the presence of Psip and Tp in this molecule. Neither of these nucleotides could be detected after hydrolysis by piperidine, NaOH, or T2 RNase. Similarly digestion with venom phosphodiesterase plus phosphatase failed to release any pseudourdine or ribothymidine. Identical results were obtained with the initiator tRNA from sheep mammary gland. The absence of these nucleosides was confirmed by pancreatic and T1 RNase digestion of the rabbit-liver initiator tRNA. The classical G-T-Psi-C sequence was not detected in this molecule. An A-U-C-G sequence has been identified; it may possibly replace the G-T-Psi-C sequence.

Using HeLa S-100 extracts, we have transcribed total DNA isolated from several species of higher animals. Transcription of the total DNA from rat, mouse, or hamster gives a discrete product of 6S RNA (about 180 nucleotides) in addition to a smeared background, whereas transcription from chick, calf, or human DNA gives a discrete band of 5S RNA (about 120 nucleotides). In view of the sensitivity to a-amanitin, these transcripts are produced by RNA polymerase III. Fingerprint analysis demonstrated that the 6S RNA species transcribed from rodent DNA have conserved sequences. The composition of dinucleotides in these bands having a guanosine residue on the 3' side was analyzed by RNase T2 digestion. The results show that the CG ratios of the transcripts of rodent, calf and chick are quite low in comparison with AG, UG, or GG except in the case of the human transcript, suggesting that these highly repetitive transcribable sequences are derived from a common prototype sequence, except in human cells. We have isolated two phage clones and one phage clone from a rat and mouse genomic library, respectively, which give a transcript of 180 nucleotides in size, and whose pattern of RNase T2 digests is the same as that of the conserved transcriptional products of total DNA. These highly repetitive transcribable sequences appear to be reiterated at several to ten thousand copies per haploid genome. The relationship of these sequences with the highly repetitive interspersed sequence B1 or Alu family is discussed.

ACTIBIND is a T2 RNase extracellular glycoprotein produced by the mould Aspergillus niger B1 (CMI CC 324626) that possesses anticarcinogenic and antiangiogenic activities. ACTIBIND was found to be an actin-binding protein that interacts with rabbit muscle actin in a 1:2 molar ratio (ACTIBIND:actin) with a binding constant of 16.17 x 10(4) M(-1). Autoclave-treated ACTIBIND (EI-ACTIBIND) lost its RNase activity, but its actin-binding ability was conserved. ACTIBIND crystals were grown using 20% PEG 3350, 0.2 M ammonium dihydrogen phosphate solution at room temperature (293 K). One to four single crystals appeared in each droplet within a few days and grew to approximate dimensions of 0.5 x 0.5 x 0.5 mm after about two weeks. Diffraction studies of these crystals at low temperature (100 K) indicated that they belong to the P3(1)21 space group, with unit-cell parameters a = 78, b = 78, c = 104 A.

The 3'-terminal structures of ribosomal 28S RNA and its precursors from rat and mouse were analyzed by means of periodate oxidation followed by reduction with 3H-borohydride. 3'-terminal labeled nucleoside derivatives produced by RNase T2 digestion were determined by thin-layer chromatography and oligonucleotides generated by RNase T1 digestion were analyzed by DEAE-Sephadex chromatography. In the rat, the major 3'-terminal sequences of ribosomal 28S RNA, nucleolar 28S, 32S, 41S, and 45S RNAs were YGUoh, GZ2Uoh, GZ12Uoh, GZ2Uoh, and GZ7Goh, respectively, whereas in the mouse corresponding sequences were YGUoh, GZ1,2, or 3Uoh, Goh, Uoh and GZ 13Uoh, respectively. (Y: pyrimidine nucleoside, Z: any nucleoside other than guanosine) These results suggest that a "transcribed spacer" sequence is present at the 3'-terminus of the 45S pre-ribosomal RNA, which is gradually removed during the steps of processing.

During in vitro synthesis of reovirus mRNA by viral cores, methyl groups from S-adenosylmethionine are incorporated only into 5'-terminal cap structures, i.e., m7GpppGmCp.... Thus, mRNA synthesized in the presence of S-adenosyl-[methyl-3H]methionine is 3H labeled specifically at the 5' terminus. This circumstance was exploited in the determination of 5'-terminal nucleotide sequences. Seven 5'-terminal fragments derived by complete RNase T1, digestion of methyl-3Hlabeled mRNA were partially degraded with RNase T2, and the products were separated by electrophoresis-homochromatography. From the patterns formed by the methyl-3H-labeled RNase T2 products, the sequences of the seven RNase T1-generated fragments were deduced. All seven fragments started with the sequence m7GpppGmCUA, after which the sequences diverged, with a tendency to be either U-rich or A-rich. Their chain lengths ranged from 7 to 10 nucleotides (excluding the m7G residue), and none of them contained an initiator AUG triplet. The sequences obtained support the hypothesis that virion-associated oligonucleotides arise through abortive transcription of the viral genome. There is no apparent 5'-terminal sequence feature distinctive of early versus late mRNA species within the small-mRNA size class.

S-RNase is associated with the gametophytic self-incompatibility of flowering plants in Solanaceae and, on the basis of sequence homology, belongs to the RNase T2 family. To identify the active site residues in S-RNase, Nicotiana alata S6-RNase was studied by chemical modification. S6-RNase was inactivated with iodoacetic acid under conditions similar to those used for the inactivation of RNase T2. No inactivation took place in the presence of 2'GMP. Analysis of carboxymethylated S6-RNase revealed that the S-carboxymethylation of Cys95 caused inactivation of the enzyme and that the two histidine residues corresponding to two essential histidine residues of RNase T2 remained intact. Treatment of S6-RNase with diethyl pyrocarbonate (DEPC) resulted in loss of enzyme activity, and the enzyme was protected from inactivation in the presence of 2'GMP. The ethoxycarbonylated residues in DEPC-inactivated S6-RNase were analyzed by mass spectrometry, which also provided structural information on sugar moieties attached to Asn27 and Asn37. His31 was modified with DEPC in the absence of 2'GMP and was not modified in its presence. His31 and His91 are conserved in all members of the RNase T2 family sequenced so far, but Cys95 is not conserved in all known Solanaceae S-RNases. These results suggest that His31, possibly together with His91, corresponding to His115 at the active site of RNase T2, is essential to the function of S6-RNase, but Cys95 is not essential though its S-carboxymethylation causes inactivation.

The active site of a base non-specific RNase from Rhizopus niveus (RNase Rh), consists of three histidine residues and one carboxyl group [Ohgi, K. et al. (1992) J. Biochem. 111, 132-138]. In order to identify this acidic amino acid residue, we chose Asp51 and Glu105 as candidates based on a comparison of the primary structures of four fungal RNases and self-incompatibility factors of Nicotiana alata which belong to the RNase T2 family. We substituted these amino acid residues with other amino acids by site-directed mutagenesis, and determined the enzymatic properties of the mutated enzymes. The enzymatic activities of E105Q, E105D, and E105A mutant enzymes were decreased markedly, but those of D51N, D51E, and D51A were decreased only slightly when RNA was used as a substrate. Therefore we concluded that Glu105 is related to the catalytic function. Kinetic constants for the enzymatic activity of E105Q and E105D toward ApU suggest that the proper size and negative charge of side chain groups are important for the catalysis of RNase Rh. However, the enzymatic activity of D51N toward ApU, but not toward UpU, decreased markedly. Therefore, we suggest that Asp51 is one of the amino acid residues forming the base recognition site. The substitution of Asp51 by Asn causes the enzyme to be more guanine nucleotide-preferential.

Ribonuclease MC1 (RNase MC1) isolated from seeds of bitter gourd (Momordica charantia) consists of 190 amino acids and is characterized by a preferential cleavage at the 5'-side of uridine. This uridine specificity distinguishes RNase MC1 from other enzymes belonging to the RNase T2 family. The three-dimensional structures of RNase MC1, in a complex with either 2'-UMP or 3'-UMP, were determined at 1.48 and 1.77 A resolutions, respectively. The side chains of Gln9 and Asn71 interact with O4 and N3, respectively, of the uracil base by hydrogen bondings. In addition, the uracil base is sandwiched by the hydrophobic side chains of Leu73 and Phe80. Compared with these amino acid residues and corresponding residues in RNases in the RNase T2 family, Gln9 and Phe80 are highly conserved in the RNases in T2 family, while Asn71 and Leu73 in RNase MC1 are variant in sequences. It is thus likely that interactions of the side chains of Asn71 and Leu73 with the uracil base are responsible for the absolute uridine specificity of RNase MC1. Site-directed mutagenesis experiments showed that replacement of Asn by Thr decreased both the catalytic efficiency and the binding affinity by 2.3- and 7.0-fold, respectively, and substitution of Leu73 for Ala predominantly decreased the binding affinity by 14. 5-fold, compared with findings in case of wild-type RNase MC1. It is thus demonstrated that Asn71 and Leu73 play an essential role in uridine preference for RNase MC1.

T2 ribonucleases are conserved nucleases that affect a variety of processes in eukaryotic cells including the regulation of self-incompatibility by S-RNases in plants, modulation of host immune cell responses by viral and schistosome T2 enzymes, and neurological development and tumor progression in humans. These roles for RNaseT2's can be due to catalytic or catalytic-independent functions of the molecule. Despite this broad importance, the features of RNaseT2 proteins that modulate catalytic and catalytic-independent functions are poorly understood. Herein, we analyze the features of Rny1 in Saccharomyces cerevisiae to determine the requirements for cleaving tRNA in vivo and for inhibiting cellular growth in a catalytic-independent manner. We demonstrate that catalytic-independent inhibition of growth is a combinatorial property of the protein and is affected by a fungal-specific C-terminal extension, the conserved catalytic core, and the presence of a signal peptide. Catalytic functions of Rny1 are independent of the C-terminal extension, are affected by many mutations in the catalytic core, and also require a signal peptide. Biochemical flotation assays reveal that in rny1Δ cells, some tRNA molecules associate with membranes suggesting that cleavage of tRNAs by Rny1 can involve either tRNA association with, or uptake into, membrane compartments.

Although rice resistance plays an important role in controlling the brown planthopper (BPH), Nilaparvata lugens, not all varieties have the same level of protection against BPH infestation. Understanding the molecular interactions in rice defense response is an important tool to help to reveal unexplained processes that underlie rice resistance to BPH. A proteomics approach was used to explore how wild type IR64 and near-isogenic rice mutants with gain and loss of resistance to BPH respond during infestation. A total of 65 proteins were found markedly altered in wild type IR64 during BPH infestation. Fifty-two proteins associated with 11 functional categories were identified using mass spectrometry. Protein abundance was less altered at 2 and 14 days after infestation (DAI) (T1, T2, respectively), whereas higher protein levels were observed at 28 DAI (T3). This trend diminished at 34 DAI (T4). Comparative analysis of IR64 with mutants showed 22 proteins that may be potentially associated with rice resistance to the brown planthopper (BPH). Ten proteins were altered in susceptible mutant (D1131) whereas abundance of 12 proteins including S-like RNase, Glyoxalase I, EFTu1 and Salt stress root protein "RS1" was differentially changed in resistant mutant (D518). S-like RNase was found in greater quantities in D518 after BPH infestation but remained unchanged in IR64 and decreased in D1131. Taken together, this study shows a noticeable level of protein abundance in the resistant mutant D518 compared to the susceptible mutant D1131 that may be involved in rendering enhanced level of resistance against BPH.

A growing number of T2/S-RNases are being discovered in plant genomes. Members of this protein family have a variety of known functions, but the vast majority are still uncharacterized. We present data and analyses of phylogenetic relationships among T2/S-RNases, and pay special attention to the group that contains the female component of the most widespread system of self-incompatibility in flowering plants. The returned emphasis on the initially identified component of this mechanism yields important conjectures about its evolutionary context. First, we find that the clade involved in self-rejection (class III) is found exclusively in core eudicots, while the remaining clades contain members from other vascular plants. Second, certain features, such as intron patterns, isoelectric point, and conserved amino acid regions, help differentiate S-RNases, which are necessary for expression of self-incompatibility, from other T2/S-RNase family members. Third, we devise and present a set of approaches to clarify new S-RNase candidates from existing genome assemblies. We use genomic features to identify putative functional and relictual S-loci in genomes of plants with unknown mechanisms of self-incompatibility. The widespread occurrence of possible relicts suggests that the loss of functional self-incompatibility may leave traces long after the fact, and that this manner of molecular fossil-like data could be an important source of information about the history and distribution of both RNase-based and other mechanisms of self-incompatibility. Finally, we release a public resource intended to aid the search for S-locus RNases, and help provide increasingly detailed information about their taxonomic distribution.

In reovirus-infected cells, virus-specific particles accumulate that have associated with them a polyribocytidylate [poly(C)]-dependent polymerase. This enzyme copies in vitro poly(C) to yield the double-stranded poly(C).polyriboguanylate [poly(G)]. The particles with poly(C)-dependent polymerase were heterogeneous in size, with most sedimenting from 300S to 550S. Exponential increase in these particles began at 23 h, and maximal amounts were present by 31 h, the time of onset of exponential growth of virus at 30 degrees C. Maximal amounts of particles with active transcriptase and replicase were present at 15 and 18 h after infection. Thereafter, there was a marked decrease in particles with active transcriptase and replicase until base line levels were reached at 31 h. Thus, the increase in poly(C)-responding particles occurred coincident with the decrease in particles with active transcriptase and replicase. The requirement for poly(C) as template was specific because no RNA was synthesized in vitro in response to any other homopolymer, including 2'-O-methyl-poly(C). Synthesis was optimal in the presence of Mn(2+) as the divalent cation, and no primer was necessary for synthesis. In contrast, the dinucleotide GpG markedly stimulated synthesis in the presence of 8 mM Mg(2+). The size of the poly(C).poly(G) synthesized in vitro was dependent on the size of the poly(C) used as template. This suggested that the whole template was copied into a complementary strand of similar size. The T(m) of the product was between 100 and 130 degrees C. Hydrolysis of the product labeled in [(32)P]GMP with alkali or RNase T2 yielded GMP as the only labeled mononucleotide. This does indicate that the synthesis of the poly(G) strand in vitro did not proceed by end addition to the poly(C) template, but proceeded on a separate strand.

The crystal structure of Escherichia coli ribonuclease I (EcRNase I) reveals an RNase T2-type fold consisting of a conserved core of six beta-strands and three alpha-helices. The overall architecture of the catalytic residues is very similar to the plant and fungal RNase T2 family members, but the perimeter surrounding the active site is characterized by structural elements specific for E. coli. In the structure of EcRNase I in complex with a substrate-mimicking decadeoxynucleotide d(CGCGATCGCG), we observe a cytosine bound in the B2 base binding site and mixed binding of thymine and guanine in the B1 base binding site. The active site residues His55, His133, and Glu129 interact with the phosphodiester linkage only through a set of water molecules. Residues forming the B2 base recognition site are well conserved among bacterial homologs and may generate limited base specificity. On the other hand, the B1 binding cleft acquires true base aspecificity by combining hydrophobic van der Waals contacts at its sides with a water-mediated hydrogen-bonding network at the bottom. This B1 base recognition site is highly variable among bacterial sequences and the observed interactions are unique to EcRNaseI and a few close relatives.

A polydnavirus, Cotesia plutellae bracovirus (CpBV), possesses segmented genome located on chromosome(s) of an endoparasitoid wasp, C. plutellae. An episomal viral segment (CpBV-S3) consists of 11,017 bp and encodes two putative open reading frames (ORFs). ORF301 shows amino acid sequence homologies (28-50%) with RNase T2s of various organisms. It also contains BEN domain in C-terminal region. ORF302 is a hypothetical gene, which is also found in other bracoviruses. Both genes were expressed in larvae of Plutella xylostella parasitized by C. plutellae. Their expressions were detected in all tested tissues including hemocyte, fat body, gut, and epidermis. To analyze effects of these genes on the parasitism, the segment of CpBV-S3 was injected to nonparasitized larvae of P. xylostella, in which the two genes were expressed at least for 4 days post-injection. The larvae injected with CpBV-S3 exhibited significant immunosuppression, such as reduction in total hemocyte population and impairment in nodule formation behavior of hemocytes in response to bacterial challenge. Each gene expression in the treated larvae was inhibited by co-injecting respective double strand RNA (dsRNA) specific to each ORF. Injection of dsRNA of ORF301 could rescue the immunosuppression of the viral segment-treated larvae, while dsRNA specific to ORF302 did not. These results suggest that a putative RNase fused with a BEN domain encoded in CpBV-S3 plays a parasitic role in inducing host immunosuppression in the parasitism.